Crop plants have a fundamental dependence on inorganic nitrogenous fertilizers, principally in the form of nitrate (NO3−) and ammonium (NH4+). Each year, approximately 85 to 90 million metric tons (MMt) of nitrogenous fertilizers are added to the soil worldwide. This amount is up from only 1.3 MMt in 1930 and from 10.2 MMt in 1960. It is predicted to increase to 240 MMt by the year 2050 (Tilman et al., 1999, Proc. Nat. Acad. Sci. USA. 96: 5995-6000). It is estimated that 50% to 70% of the applied nitrogen is lost from the plant-soil system. Because NO3− is soluble and not retained by the soil matrix, excess NO3− may leach into the water and may be depleted by microorganisms.
It is important to improve the nitrogen use efficiency (NUE) of crop plants for two reasons. First, the use of commercial fertilizers accounts for one of the major costs associated with the production of high yielding crops. Second, it would be an environmental benefit to reduce the levels of nitrogenous fertilizers that are lost into the ecosystem. Environmental effects include the deterioration of soil quality, pollution and health hazards.
Alanine is one of the more common amino acids in plants. Alanine is synthesized by the enzyme alanine aminotransferase (AlaAT) from pyruvate and glutamate in a reversible reaction, as shown in FIG. 1. Alanine is an amino acid that is known to increase under other specific environmental conditions such as drought and anaerobic stress (Muench and Good, 1994, Plant Mol. Biol. 24:417-427; Vanlerberge et al., 1993, Plant Physiol. 95:655-658). Alanine levels are known to increase substantially in root tissue under anaerobic stress. As an example, alanine levels in barley roots increase 20-fold after 24 hours of anaerobic stress. The AlaAT gene is induced by light in broom millet and when plants are recovering from nitrogen stress (Son et al., 1992, Arch. Biochem. Biophys. 289: 262-266). Vanlerberge et al. (1993) have shown that in nitrogen-starved anaerobic algae, the addition of nitrogen in the form of ammonia resulted in 93% of an N15 label being incorporated directly into alanine. Thus, alanine appears to be an important amino acid in stress response in plants.
U.S. Pat. No. 6,084,153 discloses the induction of AlaAT in the roots of canola plants and a resulting nitrogen efficient phenotype.
WO 01/55433 teaches the use of Brassica turgor gene-26 (btg26). The turgor gene-26-like proteins have recently been named antiquitins (Tang et al., 2002, FEBS Lett. 516(1-3):183-186). Brassica napus plants were transformed with constructs containing the AlaAT gene in operative linkage with the btg26 promoter. The transgenic plants were shown to have elevated levels of AlaAT in the root tissue.
US2005/0044585 discloses the use of promoters LeAMT1, LeNRT1, GmNRT2, KDC1, PHT1, GOGAT, OsRAB5 and ALF5 to direct root specific expression of a gene encoding a nitrogen utilization protein, for example, AlaAT.
Increasing NUE within rice is also desired within the art. In 2006, worldwide acreage devoted to growing rice was 151,730,000 hectares with nitrogen consumption estimated at 11,963 MMT. Thus, improving NUE in rice would not only decrease the cost of crop production but would reduce the harmful environmental effects of nitrogen fertilizers including the development of “dead zones” in the world's oceans that result from the death and decomposition of massive algae blooms fed by excessive nutrient runoff.